Embryonic stem cells (hereinafter also referred to as “ES cells”) are undifferentiated cells having pluripotency and the ability to replicate autonomously. Furthermore, it has been suggested that ES cells have the ability to repair tissues after injury. Therefore, ES cells are being vigorously studied as being useful in screening therapeutically effective substances for various diseases, and in the field of regeneration medicine. Compared to murine ES cells, simian ES cells are closer to those of humans, and therefore, they are expected to be suitable for use in human disease models.
Genetic engineering of ES cells will be extremely critical to the future application of ES cells in the treatment of various diseases and injuries. To modify ES cell properties such as drug sensitivity, the ability to proliferate and differentiate, and the like, stable gene transfer into the ES cell genome is often required. Retroviral vectors that integrate into the host genome are used routinely to achieve stable gene transfer. This is because, when cells such as ES cells that can proliferate and differentiate are used as targets, vectors will be diluted with each cell division if the introduced gene is not integrated into the genome. However, when using the retroviral vector derived from the Moloney murine leukemia virus (MOMLV), a commonly used gene transfer vector, the efficiency of gene transfer to murine ES cells is low (approximately a few percent), and the level of gene expression decreases with time. Recently, a retroviral vector derived from murine stem cell virus (MSCV) was used to improve the efficiency of gene transfer to murine ES cells (to 50% or higher). However, the problem of reduced gene expression over time has not been solved (Cherry, S. R. et al. Mol. Cell Biol. 20:7419, 2000) Recently, it was shown that the use of the lentivirus vector, another vector which can integrate into the genome, can further improve the efficiency of gene introduction into murine ES cells (to 80% or higher) (Hamaguchi, I. et al. J. Virol. 74:10778, 2000). However, in this report, expression of the introduced gene was only observed for a short time (a few days to about two weeks), and there was no record of long-term expression of the introduced gene.
Murine ES cells were used in all of the above-indicated reports of gene transfer to ES cells. To date, there have been no reports on gene transfer to primate ES cells. However, an academic meeting report indicated that gene transfer to primate ES cells is more difficult than gene transfer to murine ES cells. For example, the efficiency of gene transfer to primate ES cells has been reported to be approximately 1% using MOMLV vector, or approximately 5 to 10% using the MSCV vector (IMSUT Symposium for Stem Cell Biology, Tokyo, Japan 2000; Key Stone Sympoia, Pluripotent Stem Cells: Biology and Applications, Durango, Colo., USA, 2001).